Preliminary characterization of ferrichrome synthetase from Aspergillus quadricinctus.

An enzyme synthesizing the cyclic hexapeptide, ferrichrome, was partially purified from extracts of Aspergillus quadricinctus by fractional (NH4)2SO4 precipitation and Bio-Gel A 1.5 m filtration. About a 20-fold purification was achieved. The enzyme system incorporated delta-N-acetyl-delta-N-hydroxyornithine into ferrichrome and catalyzed ATP-PP1 exchange reactions, dependent on the constituent amino acids, glycine and delta-N-acetyl-delta-N-hydroxyornithine, in the presence of Mg2+. The optimal temperature was 27 degrees C. Km values were 3.1 . 10(-4) M for glycine and 5.3 . 10(-6) M for delta-N-acetyl-delta-N-hydroxyornithine. Both Km values were significantly lowered in the presence of 1 . 10(-6) M Fe3+. From the inhibition experiments it is concluded that sulfhydryl groups of the enzyme are involved. Both monomers are covalently bound to the enzyme in the course of the reaction. A molecular weight of 1.1 . 10(6) was determined by gel filtration. As the partially purified protein fraction also catalyzed transacetylation of hydroxyornithine from acetyl CoA, the peptide synthesizing activity may be part of a multienzyme complex. No ferrichrome synthetase activity can be found when the fungus is grown in the presence of 1. 10(-5) M Fe3+.

Purification and characterization of a nicotinamide adenine dinucleotide-dependent secondary alcohol dehydrogenase from Candida boidinii.

From the yeast Candida boidinii grown on glucose a new secondary alcohol dehydrogenase was purified 426-fold by heat treatment, column chromatography on DEAE-Sephacel, affinity chromatography on Blue Sepharose Cl-6b, and gel filtration on Sephacryl S-300. The purified enzyme was homogeneous as judged by analytical polyacrylamide gel electrophoresis. The molecular weight was found to be 150000 by sedimentation equilibrium as well as by gel filtration. The enzyme appears to be composed of four identical subunits (Mr=38000) as determined by SDS-gel electrophoresis. The enzyme catalyzes the oxidation of isopropanol to acetone in the presence of NAD+ as an electron acceptor. The Km values were found to be 0.099 mM for isopropanol and 0.14 mM for NAD+. Besides isopropanol also other secondary alcohols like butan-2-ol, pentan-2-ol, pentan-3-ol, hexan-2-ol, cyclobutanol, cyclopentanol, and cyclohexanol served as a substrate and were oxidized to the corresponding ketones. Isopropanol seems to be the best substrate for this enzyme which we therefore call isopropanol dehydrogenase. Primary alcohols are not oxidized by the enzyme. The optimum pH for enzymatic activity in the oxidation reaction was found to be 9.0, the optimal temperature is 45 degrees C. The isoelectric point of the isopropanol dehydrogenase was found to be pH 4.9. The enzyme is inactivated by mercaptide-forming reagents and chelating agents, 2-mercaptoethanol is an inhibitor. Zinc ions appear necessary for enzyme production.

Large-scale applications of NAD(P)-dependent oxidoreductases: recent developments.

NAD(P)-dependent dehydrogenases are useful catalysts for the synthesis of chiral compounds. Many active and stable enzymes are available that (with high enantioselectivity) reduce ketones or keto acids to chiral alcohols, hydroxy acids or amino acids. For economic reasons, these reactions need coupling to the simultaneous regeneration of NAD(P)H. For preparative applications, three components have to be combined: an appropriate enzyme, an efficient coenzyme-regenerating step and a suitable reaction-engineering technique.

Screening and characterization of new enzymes for biosensing and analytics.

The development of new or improved analytical methods requires new enzymes. Screening techniques utilizing enrichment cultures and rapid assay methods supported by automated or miniaturized methods are useful tools to detect new enzyme producers. Notably, oxidoreductases are well suited for analytical purposes. The NAD(P)- and oxygen-independent quinoprotein dehydrogenases with a covalently bound redox cofactor can be used advantageously for the development of biosensors. Examples are given of selective enrichment methods used in screening for useful enzyme-producing microorganisms. Enrichment under chemostatic conditions proved to be successful because enzymes with a remarkably high affinity against the analyte could be obtained. This is demonstrated by the screening of a trimethylamine-converting enzyme. The frequently observed high selectivity of these enzymes against the substrate is demonstrated in a few examples. In exploitation of these new oxidoreductases, new analytical methods were developed which are useful for the detection and during monitoring of phenylketonuria (PKU) or maple syrup urine disease (MSUD).

Simulation of measurements of uptake of 123I-iodide in the thyroid of fetal chimpanzees.

The quantitative assessment of the uptake of 123I-iodide (123I-) in the fetal thyroid in vivo was simulated in phantom measurements. First, the relationship between the depth of the fetal thyroid phantom and the two-peak ratio, the ratio of the counts in the gamma-ray and the X-ray energy windows of the registered spectrum, was determined. Subsequently, the attenuation of the gamma-ray signal in relationship to the depth of the fetal thyroid phantom was determined. Finally, the relationship between the two-peak ratio and the attenuation of the gamma-ray signal was deduced. For a reliable correlation, the signal recorded from the radioactivity surrounding the fetal thyroid phantom has to be subtracted from that obtained with the fetal thyroid phantom present. A correction curve was generated to be applied to the in vivo measurements. It is concluded that with this method determination of uptake of 123I- in the fetal thyroid is feasible.

Protection of the maternal and fetal thyroid from radioactive contamination by the administration of stable iodide during pregnancy. An experimental evaluation in chimpanzees.

The safety and efficacy of the administration of stable iodide to protect the fetal thyroid from exposure to radioactive iodide were investigated in chimpanzees in weeks 19 to 21 of pregnancy. The mean 24-h uptake of iodide in the fetal thyroid, determined with 123I-, was 1.8%. Administration of stable potassium iodide (KI), 0.65, 1.95 or 6.5 mg per kg body weight, 1 h before tracer injection reduced the fetal uptake satisfactorily. Only the higher doses were effective after 20 h. Excess iodide may impair a child's thyroid status. However, adverse effects were not found during the 11 days the animals ingested these doses. Tracer concentrations in the amniotic fluid were 30- to 130-fold lower than in the urine. The dose to the fetus from radioactivity in the maternal bladder was estimated by computer simulation. The potential increment of the risk from this dose during the ingestion of stable iodide is smaller than the reduction of risk achieved by inhibiting the uptake of radioactive iodide by the fetal thyroid. The conclusion of the experiments is that stable iodide can be used safely and effectively to protect the fetal thyroid against contamination with radioactive iodine.

Protection of the infant thyroid from radioactive contamination by the administration of stable iodide. An experimental evaluation in chimpanzees.

Protection of the thyroid from radioactive contamination by the administration of stable iodide was investigated in chimpanzees aged 2 to 98 weeks. The uptake of iodide in the thyroid was measured with 123I-. The animals were subjected to a control measurement first, and subsequently the thyroid uptake of 123I- was determined twice; once at the start and once at the end of 11 days of ingestion of 0.5, 1.5 or 5.0 mg of stable iodide per kg body weight per day. The three doses of iodide reduced the control thyroid iodide uptake of 10 to 30% to lower than 1% when ingested 1 h before exposure to the tracer and to 2-4% when ingested 20 h before exposure. In the latter experiments 0.5 mg iodide/kg was less effective than doses of 1.5 mg/kg or higher. The physiological state of the thyroid of control infant chimpanzees does not differ from that of human infants. Incidentally, an increased level of TSH was found during the ingestion of iodide, but with unaltered thyroxine levels. Therefore, it is concluded that a daily dose of 1.5 mg stable iodide/kg body weight and higher offers optimal protection of the thyroid against exposure to radioactive iodine in infants and that, when used during 10 days, it leaves the thyroid unaffected.

Current management of a donor insemination program.

Substantial evidence now exists to show that considerable maternal-fetal morbidity may result from microbiologic transmitted diseases that can be transmitted through artificial insemination by donor. In the present decade it has become increasingly clear that the use of fresh semen is potentially hazardous and its use has been discouraged by both the CDC and AFS. To minimize this risk, donor insemination programs should establish their own guidelines to thoroughly evaluate potential semen donors via history, physical examination, and laboratory evaluation before the use of donor semen after cryopreservation and quarantine. The management of a donor insemination program in the future requires uniform procedures for rigorous genetic and microbiologic screening before the selection and use of semen donors for artificial insemination.

Transient hyperprolactinemia during cycle stimulation and its influence on oocyte retrieval and fertilization rates.

Prolactin (PRL) has been shown to have inhibitory effect on follicle-stimulating hormone induced aromatase activity and estrogen biosynthesis in human granulosa cells cultured in vitro. To investigate the validity of the hypothesis that transient hyperprolactinemia during controlled ovarian hyperstimulation might influence follicular steroidogenesis and oocyte maturation, we measured serum PRL, estradiol, and progesterone before aspiration of oocytes in women undergoing ovarian stimulation (n = 108) in in vitro fertilization-embryo transfer. No correlation was detected between PRL and total number oocytes, number mature oocytes, fertilization rate, cleavage rate, and pregnancy rate. Transient elevation of PRL was a common finding in patients (57%) but was not associated with a poor clinical outcome.

Neonatal screening for maple syrup urine disease by an enzyme-mediated colorimetric method.

A microplate-based, enzyme-mediated, colorimetric method using L-leucine dehydrogenase (EC 1.4.1.9) has been developed for the determination of the combined branched-chain amino acids in plasma and blood-spot specimens. The test exhibits acceptable precision and fits into a new concept according to which the different parameters of neonatal screening programs for metabolic disorders, such as phenylalanine (phenylketonuria), galactose/galactose-1-phosphate (galactosemia) and branched-chain amino acids (maple syrup urine disease) can be measured in the same blood-spot eluate by use of different specific NAD(H)-dependent enzymes.

A convenient enzymatic method for the determination of 4-methyl-2-oxopentanoate in plasma: comparison with high performance liquid chromatographic analysis.

A simple and rapid spectrophotometric method for the estimation of 4-methyl-2-oxopentanoate in plasma samples by use of NAD+-dependent D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei ssp. pseudoplantarum is described. It is based on the kinetic measurement of the decrease of NADH absorbance at 334 nm. Applicability is demonstrated by comparative measurement of 4-methyl-2-oxopentanoate content in plasma of patients with maple syrup urine disease by the enzymatic and a reversed phase high performance liquid chromatographic method.

A new approach to the newborn screening for hyperphenylalaninemias: use of L-phenylalanine dehydrogenase and microtiter plates.

We adapted the recently described colorimetric method for the specific determination of phenylalanine to a microplate assay using a NAD(H)-dependent L-phenylalanine dehydrogenase. With respect to sensitivity, analytical recovery and interrun imprecision this method for measuring phenylalanine in eluates of paper-dried blood spots is suitable for routine newborn screening for hyperphenylalaninemias. In contrast to the microbiological Guthrie assay, with the enzymatic method quantitative data may be obtained on the same day, also in the blood of newborns on antibiotic treatment.

Determination of (S)- and (R)-2-oxo-3-methylvaleric acid in plasma of patients with maple syrup urine disease.

An enzymatic method for the separate measurement of both chiral 2-oxo-3-methylvaleric acid (OMV) compounds, (S)- and (R)-OMV, by NADH-dependent enantioselective amination using leucine dehydrogenase in the presence of a NADH regenerating system is described. This method allows the quantitative determination of all branched-chain 2-oxo acids, simultaneously. In plasma samples from classical maple syrup urine disease patients under therapy the average (R)-OMV/(S)-OMV ratio was 0.35 and great differences in the transamination equilibria of the diastereomeric branched-chain amino acids L-isoleucine and L-alloisoleucine were demonstrated.

Suppression of LH-stimulated prostaglandin and progesterone accumulation in rat granulosa cells by isoquinolinesulfonamide protein kinase inhibitors.

Rat granulosa cells were incubated with isoquinolinesulfonamide inhibitors of protein kinases A and C and/or LH, dibutyryl cAMP (dbcAMP), tetradecanoylphorbol acetate (TPA), cholera toxin, or forskolin for 5 h. H7 (25 microM) was observed to inhibit LH, cholera toxin or dbcAMP stimulation of prostaglandin (PGE), and progesterone accumulation. H7 produced inhibition when added as little as 2 min before and as long as 1 h after LH. HA1004 was ineffective against LH or cholera toxin stimulation of PGE or progesterone at up to 100 microM. H9 blocked some LH and forskolin responses at 25 microM, but required a 50 microM concentration to minimally affect TPA stimulation. Cytotoxicity was not observed at the concentrations and times of isoquinolinesulfonamides tested. H7 and H9, therefore, suppress LH stimulation of granulosa cell functions in a dose- and time-dependent manner consistent with inhibition of protein kinases A and/or C, and consonant with a requirement for such kinases in LH action.

Modern medical management of endometriosis.

The modern medical management of endometriosis has changed considerably since the first attempts were made to control this disease hormonally over four decades ago. Currently, there are multiple choices for the clinician and patient, including oral contraceptives, danazol, GnRH agonist analogues, and gestrinone. Several advances have been made in the use of GnRH agonists in preventing some of the untoward effects of prolonged hypoestrogenism. These add-back regimens provide the best therapy available today for prolonged medical control of endometriotic symptoms. The antiprogesterones (RU-486) hold promise for the future, but are still in the investigational stage of development.

PCDOSE: an interactive software system to calculate internal radiation dose on a personal computer.

An interactive, menu directed, software system to calculate committed dose equivalents for individuals with different physiques after inhalation, ingestion or injection of radionuclides has been developed. The calculations are based on ICRP 26/30 methods. The programs are written in PASCAL and can be implemented on a personal computer with a MS-DOS operating system and a hard disk with a storage capacity of at least 20 Mb. This paper describes the development and features of the system.

[A new procedure for the direct demonstration of microbial aminoacylase on agar plates with o-phthalaldehyde]. / Ein neues Verfahren zum direkten Nachweis mikrobieller Aminoacylaseaktivität auf Agarplatten mit o-Phthalaldehyd.

Not less than 1 nmol of methionine and phenylalanine were detected as fluorescent spots on agar plates with o-phthalaldehyde reagent under UV light. Microorganisms were grown on thin-agar-layer coated filter papers placed on nutrient agar plates, and then transferred onto new plates lacking nutrients by moving the papers. After the background amino compounds were removed by the diffusion to the bottom plates, the paper cultures were moved and incubated on assay plates containing N-acetylmethionine or N-acetylphenylalanine. The amino acids formed around the colonies were visualized by the o-phthaladehyde procedure to indicate aminoacylase activity of microorganisms. The substrate-and stereospecificity of the enzyme was shown for some strains on the agar plates by this procedure.

Sorption behaviour of Am on marl groundwater colloids.

The sorption of americium on marl colloids is studied in a comprehensive way. In Na-HCO3 groundwater conditions, americium in the concentration range 3 x 10(-11)-3 x 10(10) M sorbs onto groundwater colloids. In these tests, the colloid concentration is rather high, i.e. Ccoll 1.7+/-1.3 mg l(-1) for sizes 505000 nm or Ncoll (1.7+/-0.1) x 10(11) l(-1) for sizes larger than 100 nm. These colloids are mainly chlorite and smectite/illite with an average size smaller than 500 nm. For the groundwater (pH 8.6, [Na+]-[HCO3]-2 x 10(-2) M), Kd values in the range 1-3 x 10(6)mlg(-1) are measured. These values are compared with experimental and modelled values reported earlier in the literature.